CEA and cellular mucin antigens have been recognized as potential targets for specific immunotherapy and are frequently expressed in bladder cancer. We studied the coordinated expression of a bladder cancer-associated CEA glycoform and of the mucins MUC1, MUC2 and MAUB under various growth conditions in the MGH-U3 bladder-cancer cell line. CEA and MUC2 mRNAs and proteins were detected in nude mouse tumors and spheroids but not in monolayer cultures. Expression of MAUB and bladder-cancer CEA also was induced according to spatial configuration of cells. MUC1 was always expressed under various growth conditions, but its glycosylation was modulated: in spheroids and mostly in tumor cells, the SM3 protein epitope was unmasked and sialyl-Tn was induced. The kinetics of modulation of MAUB and bladder-cancer CEA were different. The epitope recognized by the monoclonal antibody (MAb) 19A211 was rapidly induced in the aggregation phase of spheroid formation and rapidly lost upon plating of tumor cells, suggesting a relationship with cell contact. By contrast, MAUB induction in spheroids was delayed to the compaction phase, when cell aggregates become resistant to disruption, and loss of expression upon tumor plating occurred slowly over several culture passages. No induction of these 2 antigens was observed in the presence of differentiation agents, endothelial cell products or interferon-gamma, but it occurred when MGH-U3 cells were cultured at high density on extracellular matrix. Our results suggest that CEA and mucin antigen expression in bladder cancer is modulated by the spatial configuration of cells.